Research Summary Metabolic networks in biological systems are functionally interconnected and mutually complementary, such that malfunctions in one network are compensated for by other networks, a process termed adaptive metabolism. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and utilizes this strategy to form persisters, a phenotypic variant that survives under external stresses such as antibiotics, thus contributing to species conservation. The ability of Mtb to undergo adaptive metabolism, thus ensuring persistence in the host and maximizing tolerance against host environmental stresses, is responsible for the prolonged nature of standard TB chemotherapies. Therefore, new therapeutic options that prevent adaptive metabolism and subsequent persister formation are urgently needed, but scientific knowledge regarding persister formation in Mtb is scant due to lack of in vitro persister reconstitution systems and optimal analytical techniques. Here, we seek to overcome these hurdles by incorporating an in vitro biofilm system, FACS, and cutting-edge metabolomics. Our new methodologies will enable us to pinpoint the adaptive metabolic mechanism specific to Mtb persisters. We?ve shown that Mtb persisters downregulate electron transport chain (ETC) activity to reduce the production of deleterious reactive radicals, meanwhile, activate adaptive metabolism to correct malfunctional networks due to the absence of canonical ETC activity. Using our in vitro biofilm/FACS method, we selectively isolated Mtb persisters and revealed that altered trehalose metabolism compensated for a decrease in ETC activity, but Mtb deficient in trehalose metabolism are still able to form persisters at a density similar to that of wild type, albeit after a significant initial delay. Collectively, these data indicate that Mtb is equipped with multilayered adaptive strategies for persister formation. We will use our new method to selectively isolate persisters derived from wild type Mtb or trehalose metabolism deficient Mtb to delineate trehalose metabolism I. mediated and II. independent adaptive activities. Once the functional essentiality of I and II for persister formation are validated, we will exploit the relative contribution of I and/or II for metabolic adaptive activities to drug- tolerance. This project will significantly advance our insight into the ability of Mtb to adapt to antibiotic stresses and hostile host environments. We anticipate the identification and exploration of new drug targets to improve our control over the TB pandemic.